Hanazawa M, Mochii M, Ueno N, Kohara Y, Iino Y
Molecular Genetics Research Laboratory, University of Tokyo, Tokyo 113-0033, Japan.
Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8686-91. doi: 10.1073/pnas.141004698. Epub 2001 Jul 10.
Caenorhabditis elegans is an ideal organism for the study of the molecular basis of fundamental biological processes such as germ-line development, especially because of availability of the whole genome sequence and applicability of the RNA interference (RNAi) technique. To identify genes involved in germ-line development, we produced subtracted cDNA pools either enriched for or deprived of the cDNAs from germ-line tissues. We then performed differential hybridization on the high-density cDNA grid, on which about 7,600 nonoverlapping expressed sequence tag (EST) clones were spotted, to identify a set of genes specifically expressed in the germ line. One hundred and sixty-eight clones were then tested with the RNAi technique. Of these, 15 clones showed sterility with a variety of defects in germ-line development. Seven of them led to the production of unfertilized eggs, because of defects in spermatogenesis (4 clones), or defects in the oocytes (3 clones). The other 8 clones led to failure of oogenesis. These failures were caused by germ-line proliferation defect (Glp phenotype), meiotic arrest, and defects in sperm--oocyte switch (Mog phenotype) among others. These results demonstrate the efficacy of the screening strategy using the EST library combined with the RNAi technique in C. elegans.
秀丽隐杆线虫是研究生殖系发育等基本生物学过程分子基础的理想生物，尤其是因为其全基因组序列的可获得性以及RNA干扰（RNAi）技术的适用性。为了鉴定参与生殖系发育的基因，我们制备了富集或缺失生殖系组织cDNA的消减cDNA文库。然后我们在高密度cDNA微阵列上进行差异杂交，该微阵列上点样了约7600个非重叠表达序列标签（EST）克隆，以鉴定一组在生殖系中特异性表达的基因。然后用RNAi技术对168个克隆进行了检测。其中，15个克隆表现出不育，生殖系发育存在各种缺陷。其中7个导致未受精卵的产生，原因是精子发生缺陷（4个克隆）或卵母细胞缺陷（3个克隆）。另外8个克隆导致卵子发生失败。这些失败是由生殖系增殖缺陷（Glp表型）、减数分裂停滞以及精子-卵母细胞转换缺陷（Mog表型）等引起的。这些结果证明了在秀丽隐杆线虫中使用EST文库结合RNAi技术的筛选策略的有效性。
